Conventional antisera, produced by immunizing animals with tumor cells or other antigens, contain a myriad of different antibodies differing in their specificity and properties. In 1975 Kohler and Milstein (Nature, 256:495) introduced a procedure which leads to the production of quantities of antibodies of precise and reproducible specificity. The Kohler-Milstein procedure involves the fusion of spleen cells (from an immunized animal) with an immortal myeloma cell line. By antibody testing of the fused cells (hybridomas), clones of the hybridomas are selected that produce antibody of the desired specificity. Each clone continues to produce only that one antibody, monoclonal antibody (mAb). As hybrodoma cells can be cultured indefinitely (or stored frozen in liquid nitrogen), a constant, adequate supply of antibody with uniform characteristics is assured.
Antibodies are proteins that have the ability to combined with and recognize other molecules, known as antigens. Monoclonal antibodies are no different from other antibodies except that they are very uniform in their properties and recognize only one antigen or a portion of an antigen known as a determinant.
In the case of cells, the determinant recognized is an antigen on or in the cell which reacts with the antibody. It is through these cell antigens that a particular antibody recognizes, i.e. reacts with, a particular kind of cell. Thus the cell antigens are markers by which the cell is identified.
These antigenic markers may be used to observe the normal process of cell differentiation and to locate abnormalities within a given cell system. The process of differentiation is accompanied by changes in the cell surface antigenic phenotype, and antigens that distinguish cells belonging to distinct differentiation lineages or distinguish cells at different phases in the same differentiation lineage may be observed if the correct antibody is available.
The preparation of hybridoma cell lines can be successful or not depending on such experimental factors as nature of the innoculant, cell growth conditions, hybridization conditions etc. Thus it is not always possible to predict successful hybridoma preparation of one cell line although success may have been achieved with another cell line.
Process in defining cell surface antigens is of great importance in differentiation and disease as markers for normal and diseased cells, thereby furthering diagnosis and treatment. Thus work on melanocytes was made possible by the recently discovered technique of culturing melanocytes from normal skin (Eisinger, et al., Proc. Nat'l. Acad. Sci. U.S.A., 79 2018 (Mar. 1982). This method provides a renewable source of proliferating cells for the analysis of melanocyte differentiation antigens. Likewise, a large number of cell lines derived from melanomas have now been established and these have facilitated the analysis of melanoma surface antigens. The advent of mAbs has greatly accelerated knowledge about the surface antigens of malignant melanoma. cell markers on both melanomas and melanocytes have been identified. A panel of typing monoclonal antibodies has been selected which recognizes differentiation antigen characteristics at each stage of development in both melanocytes and melanomas. These differentiation antigens may be used to classify melanocytes and melanomas and to group them into characteristic sub-sets. [Dippold et al. Proc. Nat'l. Acad. Sci. U.S.A. 77, 6114 (1980) and Houghton, et al. J. Exp. Med. 156, 1755 (1982)]. Immunoassy of melanocytes and melanoma cells within sub-sets is thus made possible.
Initial recognition of differentiation antigens came about through analysis of surface antigens of T-cell leukemias of the mouse and the description of the TL, Thy-1, and Lyt series of antigens. (Old, Lloyd J., Cancer Research, 41, 361-375, Feb. 1981) The analysis of these T-cell differentiation antigens was greatly simplified by the availability of normal T cells and B cells of mouse and man. (See Patents #4,361,549-559; #4,3643,932-37 and #4,363,799 concerning mAb to Human T-cell antigens).
The existence of human leukemia specific antigens has been suggested by studies using heterologous antibodies developed by immunization with human leukemic cells [Greaves, M. F. et al. Clin. Immunol. and Immunopathol 4:67, (1975); Minowada, J., et al. J. Nat'l. Cancer Insti. 60:1269, (1978); Tanigaki, N., et al. J. Immunol. 123:2906, (1979)] or by using autologous antisera obtained from patients with leukemia [Garret, T. J., et al., Proc. Nat'l. Acad. Sci. U.S.A. 74:4587, (1977); Naito, K., et al., Proc. Nat'l. Acad. Sci. U.S.A., 80: 2341, (1983)]. The common acute lymphoblastic leukemia antigen (CALLA) which is present on leukemia cells from many patients with non-T, non-B, acute lymphoblastic leukemia (N-ALL), some chronic myelocytic leukemias (CML) in blast crisis and a few acute T-lymphoblastic leukemias (T-ALL) was originally described using conventional rabbit heteroantisera [Greaves, M. F. et al. Supra]. By the autologous typing technique [Garret, T. J., et al. Supra; Naito, K., et al. Supra 1983; Old, L. J. Cancer Res. 41:361, (1981)], antibodies uniquely reacting with ALL cells were found in sera obtained from patients with ALL, and seemed to recognize very similar antigens to CALLA (Garret, T. J., et al. Supra; Naito, K., et al. Supra). Another leukemia associated antigen detected by heterologous antisera is the human thymus leukemia (TL)-like antigen, which is present on thymocytes as well as leukemia cells (Tanigaki, N. et al. Supra). This antigen, is therefore, a normal differentiation antigen which is composed of a heavy chain (MW 44,000-49,000 ) and light chain (MW 12,000-14,000) similar to the class I HLA antigens (Tanigaki, N., et al. Supra). These investigations have, however, been hampered by the need for vigorous absorptions with normal tissues as well as the relatively small quantity and low titer of the antisera.
In vitro production of monoclonal antibodies by the technique of Kohler and Milstein, Supra has provided a better system for the identification and detection of leukemia specific antigens. A panel of monoclonal antibodies detecting cell surface antigens of human peripheral blood lymphocytes and their precursor cells have been investigated in detail [Reinherz, E. L., et al. Proc. Nat'l. Acad. Sci. U.S.A. 77:1588, (1980)]. While monoclonal antibodies detecting antigens characteristic for different lymphocyte lineages can be used for classification of human lymphocytic leukemia [Schroff, R. W., et al. Blood 59: 207, (1982)], such antibodies have only limited therapeutic applications. Monoclonal antibodies detecting human leukemia associated antigens have also been produced. These include several antibodies detecting the human equivalents of the murine TL antigens. One TL-like antigen is recognized by Na1/34 [McMichael, A. J. , et al. Eur. J. Immunol. 9:205, (1979)], OKT6 (Reinherz, E. L., et al. Supra) and Leu 6 (R. Evans, personal communication). A second TL-like antigen is recognized by M241 (Knowles, R. W., et al. Eur. J. Immunol. 12: 676,1982). Monoclonal antibodies with specificities for common acute lymphoblastic leukemia antigens J-5 (Ritz, J., et al. Nature 283:583, 1980), NL-1 and NL-22 (Ueda, R., et al. Proc. Nat'l. Acad. Sci. U.S.A. 79:4386, 1982) have also been produced. Recently, Deng, C-T, et al. Lancet. i:10, 1982) reported a complement fixing monoclonal antibody (CALLA-2) which reacts with most cultured human T-ALL cell lines and also reacts with most fresh T-ALL cells.
Mouse monoclonal antibodies to human tumor cell surface antigens have been produced in many laboratories (Lloyd, K. O. (1983) In: Basic and Clinical tumor Immunology, Vol. 1 (R. B. Herberman, Ec.), Nijhoff, The Hague (in press)). The intention of these studies often has been to identify tumor-associated antigens that could be useful in tumor therapy or diagnosis. An inherent difficulty in this approach is the diversity of antigens on the cell surface. Although it has been possible to identify some antigens with a very restricted distribution, antibodies to antigens that elicity very weak immune responses may be missed due to their scarcity. Also, immunization with a complex mixture of antigens such as tumor cells may suppress the antibody response to relatively less immunogenic molecules, in a manner resembling antigenic competition (Taussig, M. J. (1973). Curr. Top. Micro. Immuno. 60:125). One approach to circumvent these difficulties is to prepare antibodies to the strongly immunogenic antigens and to use them to remove those antigens from subsequent immunogens; this approach has been designated the "cascade" procedure (Springer, T. A. (1981) J. Biol. Chem. 256:3833) since it allows the generation of new antibodies, different from those previously obtained. The present invention provides for cancer diagnosis and therapy and overcomes problems heretofor encountered in the prior art.